Sitios de Fenoles

Introduction:

The calculation of solvent site clusters allows to generate information related to the area, time, and type of interactions. That said, we present a brief tutorial for calculating solvent sites using the results of the molecular dynamics run with mixed solvents as input information.

Pipeline Summary

Header Step	Description	Input	Output	Note
Reference	PDB reference provides	receptor_ref.pdb	X
Dynamics	Run molecular dynamics with phenols.	receptor_ref.pdb receptor.nc receptor.prmtop	Trajectory, parameters and reference files.	Save the values of trajectory time and total frames.
Phenol Sites	Calculate phenol clusters with molecular dynamics results.	receptor.prmtop receptor.nc receptor_ref.pdb parameters.in run_sugar.sh sugar-gitlab-directory	Molsites, overlaps and restime.	VMD has to be installed to run the cluster scripts.

Note: It’s important to have VMD installed. You can download all necessary files by clicking here.

Running sugar in the command line step by step

Untar the hotspots.tar.bz2 file:

yourpath:$ tar -xf hotspots.tar.bz2 -C hotspots/

You will get the following files:

inputs/

1. • 4mrhA_phe-opc_HMR-noWAT.prmtop
   • MD-4mrhA_phe-opc_HMR-1us-FEN-noWAT.nc
   • 4mrhA_ref.pdb
   • parameters.in
   • run_sugar.sh
2. outputs/
   • cd44_10_14_FEN_DU/
   • molsites/
     • SS_Res_AT.pdb
   • overlaps/
     • overlap_Res_AT.pdb
     • temp_Res_AT.pdb
     • rmsd_fit.csv
   • restime/
     • SS_#_Res_AT.site
3. references/
   • 5SC5-A.pdb
4. sugar_clusters_tcl/
   • main.tcl
   • overlap.tcl
   • parameters_example.in
   • parser.tcl
   • pru_main.tcl
   • qt_clustering.tcl
   • README.md
   • residence_time.tcl
   • solvent.tcl
   • sugar_driver.slurm.sh

You can open the parameter.in file and write the parameters of your choice.
FOLDNAME	= cd44_10_14_FEN_DU         # Folder Name
TRAJECTORY	= MD-4mrhA_phe-opc_HMR-1us-FEN-noWAT.nc         #  FILE( *.nc or *.binpos)
TOPOLOGY	= 4mrhA_phe-opc_HMR-noWAT.prmtop         #  FILE (*.parm7 or *.prmtop)
REFERENCE	= 4mrhA_ref.pdb         #  FILE (*.pdb)
STEP		= 10         #  Indicates how often you want  calculate the cluster. Its expressed in frames per nanosecond
CLUSTER_RADIUS = 1.4         #  Indicates the radius from the dummy atom center in which the phenols are found, Armstrongs (float)                                                                                                          
BINDING_SITE = resid 1 to 156         #  Binding Site (atomselect VMD class)
SOLVENT	= 	FALSE         #  Keep and run the algorithm for solvent and cosolvent if TRUE. If FALSE only runs cosolvent                                                 
SOLVENT_THRESHOLD= 0.1         #  Lowest threshold to build pre-clusters.
COSOLVENT	= 	FEN         #  atomselect VMD class
CS_ATOM_PROBE= 	DU,O1         #  atomselect VMD class
COSOLVENT_THRESHOLD= 0.2         #  Lowest threshold to build pre clusters.
N_CUT_RATIO	= 	0.1         #   Percentage of the MD time where the phenol is found. e.g. 0.1 = 10% of the time.

Check the path in the next files:

1. /inputs/run_sugar.sh
2. vmd
3. hotspot directory path e.g yourpath/hotspots/

You can open the run_sugar.sh file and write the path of your choice. Usually, you should change the path where you have installed VMD and where you saved the sugar_clusters directory, they are highlighted in bold.

#!/bin/bash
rm sugar.out
time vmd  -dispdev text -e yourpath/sugar_clusters_tcl/ main.tcl -args $@ > sugar.out 2>&1

sugar_clusters_tcl/main.tcl set dir ”yourpath/hotspots/sugar_clusters_tcl” You can open the main.tcl file and write your working directory path as highlighted in bold.

1. set dir [lindex $argv 2]

set dir "yourpath/hotspots/sugar_clusters_tcl"

Run the script: yourpath/hotspots/inputs/$ ./run_sugar.sh

Note: You can check the run status by opening the sugar.out file.

Open results and references in VMD. Go to your outputs directory. There you will find the following files:

1. Outputs

References 5SC5-A.pdb 4mrhA_ref.pdb cd44_10_14_FEN_DU Molsites SS_Res_AT.pdb SS_Res_AT.csv Overlaps overlap_Res_AT.pdb temp_Res_AT.pdb rmsd_fit.csv Restime SS_#_Res_AT.site

Open VMD to analyze the results.

yourpath/hotspots/outputs/$ vmd -m receptor_ref.pdb 5SC5-A.pdb SS_Res_AT.pdb

Set the representations to analyze the results.

Representations > 4mrhA_ref.pdb > draw > newcartoon
Representations > 5SC5-A.pdb > draw > newcartoon
Representations > 5SC5-A.pdb > draw > liicorice
Representations > 5SC5-A.pdb > selection >resame ZJY HOH
Representations > SS_Res_AT.pdb > draw > VDW
Representations > SS_Res_AT.pdb > color > Beta

1. Tutorial

Molecular dynamics

Save the parameters used during the MD simulations.

Parameter Value Note Biomolecule Protein Type proteinmask e.g. 1-1446 It depends on the system solventmask WAT,FEN Solvent Mask (WAT or WAT, ETA or WAT, FEN) timemd 10,100, 1000… MD (t) Nanoseconds in total framexns 10,20, 50, 500… Frames per nanosecond

1. Run the molecular dynamic simulations and save the following files:

File Content Note receptor.prmtop Parameter file

• .prmtop or *.parm7

receptor_ref.pdb Reference file

• .pdb

receptor.nc Trajectory file

• .nc or *.binpos

Solvent clusters. Put the previous and the following files into the same directory:

Software/script File ¿What is it? Command Note VMD VMD Visual Molecular Dynamics $ vmd Change the path onto sugar_run.sh Script parameters.in Script with parameters in tcl language to calculate clusters $ vi parameters.in It takes the *.prmtop, reference and trajectory files as input. Has to be in the same directory as the MD results Script run_sugar.sh Script to call VMD and to run the main.tcl script $ ./run_sugar.sh Has the instructions to find the clusters Has to be in the same directory as the MD results

You have to have a directory with all necessary files to run the sugar cluster script.

Running phenol cluster algorithm: Fill the file parameter.in as follow:

Command Input Note FOLDNAME = folder_name Name of directory where results will be stored TRAJECTORY = receptor_cosolvent_results.nc FILE( *.nc or *.binpos) TOPOLOGY = parameters_file.prmtop FILE (*.parm7 or*.prmtop) REFERENCE = receptor_ref.pdb FILE (*.pdb) STEP = 10 step We need to know how many frames we have. total frames/STEP. CLUSTER_RADIUS = 0.6 FLOAT BINDING_SITE = resid 44 46 48 55 57 64 67 Binding Site (atomselect vmd class) SOLVENT = TRUE

SOLVENT_THRESHOLD = 0.1 Inferior edge for pre-clusters COSOLVENT = ETA atomselect vmd class CS_ATOM_PROBE = O1, H1, C1 atomselect vmd class COSOLVENT_THRESHOLD = 0.2

N_CUT_RATIO = 0.05 porcentaje de corte para armar los precluster

Run the job $ ./run_ugar.sh

Visualization and analysis. Once the run is finished a directory with name “folder_name” will be created at the work directory. Inside will be 3 new folders named Molsites, Overlaps, and Restime.

Directory File Content Note Work directory sugar.out Info about the run

Molsites SS_Res_AT.pdb

File with Clusters info

Res= resid AT = atom type

1. = cluster number

Molsites SS_Res_AT.csv

Site with cluster info

Overlaps overlap_Res_AT.pdb

File with Overlap info

Overlaps temp_Res_AT.pdb

File with Res time info

Overlaps rmsd_fit.csv File with rmsd dit info Restime SS_#_Res_AT.site File with restime per cluster info

You will get the following files:

inputs/ 4mrhA_phe-opc_HMR-noWAT.prmtop MD-4mrhA_phe-opc_HMR-1us-FEN-noWAT.nc 4mrhA_ref.pdb parameters.in run_sugar.sh outputs/ cd44_10_14_FEN_DU/ molsites/ SS_Res_AT.pdb overlaps/ overlap_Res_AT.pdb temp_Res_AT.pdb rmsd_fit.csv restime/ SS_#_Res_AT.site references/ 5SC5-A.pdb sugar_clusters_tcl/ main.tcl overlap.tcl parameters_example.in parser.tcl pru_main.tcl qt_clustering.tcl README.md residence_time.tcl solvent.tcl sugar_driver.slurm.sh

You can open the parameter.in file and write the parameters of your choice.

FOLDNAME = cd44_10_14_FEN_DU # Folder Name TRAJECTORY = MD-4mrhA_phe-opc_HMR-1us-FEN-noWAT.nc # FILE( *.nc or *.binpos) TOPOLOGY = 4mrhA_phe-opc_HMR-noWAT.prmtop # FILE (*.parm7 or *.prmtop) REFERENCE = 4mrhA_ref.pdb # FILE (*.pdb) STEP = 10 # Indicates how often you want calculate

                                                                                                         the cluster. Its expressed in frames per
                                                                                                         nanosecond

CLUSTER_RADIUS= 1.4 # Indicates the radius from the dummy

                                                                                                         atom center in which the phenols are
                                                                                                         found, Armstrongs (float)

BINDING_SITE = resid 1 to 156 # Binding Site (atomselect VMD class) SOLVENT = FALSE # Keep and run the algorithm for solvent

                                                                                                         and cosolvent if TRUE. If FALSE only 
                                                                                                         runs cosolvent

SOLVENT_THRESHOLD= 0.1 # Lowest threshold to build pre-clusters. COSOLVENT = FEN # atomselect VMD class CS_ATOM_PROBE= DU,O1 # atomselect VMD class COSOLVENT_THRESHOLD= 0.2 # Lowest threshold to build pre clusters. N_CUT_RATIO = 0.1 # Percentage of the MD time where

                                                                                                         the phenol is found. e.g. 0.1 = 10% of
                                                                                                         the time.

Check the path in the next files: /inputs/run_sugar.sh vmd hotspot directory path e.g yourpath/hotspots/

You can open the run_sugar.sh file and write the path of your choice. Usually, you should change the path where you have installed VMD and where you saved the sugar_clusters directory, they are highlighted in bold.

1. !/bin/bash

rm sugar.out time vmd -dispdev text -e yourpath/sugar_clusters_tcl/ main.tcl -args $@ > sugar.out 2>&1

sugar_clusters_tcl/main.tcl set dir ”yourpath/hotspots/sugar_clusters_tcl” You can open the main.tcl file and write your working directory path as highlighted in bold.

1. set dir [lindex $argv 2]

set dir "yourpath/hotspots/sugar_clusters_tcl"

Run the script: yourpath/hotspots/inputs/$ ./run_sugar.sh

Note: You can check the run status by opening the sugar.out file.

Open results and references in VMD. Go to your outputs directory. There you will find the following files: Outputs References 5SC5-A.pdb 4mrhA_ref.pdb cd44_10_14_FEN_DU Molsites SS_Res_AT.pdb SS_Res_AT.csv Overlaps overlap_Res_AT.pdb temp_Res_AT.pdb rmsd_fit.csv Restime SS_#_Res_AT.site

Open VMD to analyze the results.

yourpath/hotspots/outputs/$ vmd -m receptor_ref.pdb 5SC5-A.pdb SS_Res_AT.pdb

Set the representations to analyze the results.

Representations > 4mrhA_ref.pdb > draw > newcartoon Representations > 5SC5-A.pdb > draw > newcartoon Representations > 5SC5-A.pdb > draw > liicorice Representations > 5SC5-A.pdb > selection >resame ZJY HOH Representations > SS_Res_AT.pdb > draw > VDW Representations > SS_Res_AT.pdb > color > Beta

Beta color code in VMD.

Highest scored site Middle scored site Lowest scored site

Tutorial

Molecular dynamics

Save the parameters used during the MD simulations.

Parameter Value Note Biomolecule Protein Type proteinmask e.g. 1-1446 It depends on the system solventmask WAT,FEN Solvent Mask (WAT or WAT, ETA or WAT, FEN) timemd 10,100, 1000… MD (t) Nanoseconds in total framexns 10,20, 50, 500… Frames per nanosecond

Run the molecular dynamic simulations and save the following files:

File Content Note receptor.prmtop Parameter file

• .prmtop or *.parm7

receptor_ref.pdb Reference file

• .pdb

receptor.nc Trajectory file

• .nc or *.binpos

Solvent clusters. Put the previous and the following files into the same directory:

Software/script File ¿What is it? Command Note VMD VMD Visual Molecular Dynamics $ vmd Change the path onto sugar_run.sh Script parameters.in Script with parameters in tcl language to calculate clusters $ vi parameters.in It takes the *.prmtop, reference and trajectory files as input. Has to be in the same directory as the MD results Script run_sugar.sh Script to call VMD and to run the main.tcl script $ ./run_sugar.sh Has the instructions to find the clusters Has to be in the same directory as the MD results

You have to have a directory with all necessary files to run the sugar cluster script.

Running phenol cluster algorithm: Fill the file parameter.in as follow:

Command Input Note FOLDNAME = folder_name Name of directory where results will be stored TRAJECTORY = receptor_cosolvent_results.nc FILE( *.nc or *.binpos) TOPOLOGY = parameters_file.prmtop FILE (*.parm7 or*.prmtop) REFERENCE = receptor_ref.pdb FILE (*.pdb) STEP = 10 step We need to know how many frames we have. total frames/STEP. CLUSTER_RADIUS = 0.6 FLOAT BINDING_SITE = resid 44 46 48 55 57 64 67 Binding Site (atomselect vmd class) SOLVENT = TRUE

SOLVENT_THRESHOLD = 0.1 Inferior edge for pre-clusters COSOLVENT = ETA atomselect vmd class CS_ATOM_PROBE = O1, H1, C1 atomselect vmd class COSOLVENT_THRESHOLD = 0.2

N_CUT_RATIO = 0.05 porcentaje de corte para armar los precluster

Run the job $ ./run_ugar.sh

Visualization and analysis. Once the run is finished a directory with name “folder_name” will be created at the work directory. Inside will be 3 new folders named Molsites, Overlaps, and Restime.

Directory File Content Note Work directory sugar.out Info about the run

Molsites SS_Res_AT.pdb

File with Clusters info

Res= resid AT = atom type

1. = cluster number

Molsites SS_Res_AT.csv

Site with cluster info

Overlaps overlap_Res_AT.pdb

File with Overlap info

Overlaps temp_Res_AT.pdb

File with Res time info

Overlaps rmsd_fit.csv File with rmsd dit info Restime SS_#_Res_AT.site File with restime per cluster info